Exposure of skin to ultraviolet radiation (UVR) induces formation of free radicals and oxidants (singlet oxygen, hydroxy radical, hydrogen peroxide, peroxynitrite, superoxide anions, etc.) collectively referred to as reactive oxygen species (ROS) (Hanson K M, Clegg R M. Photochemistry and Photobiology, 2002, 76(1): 57-63; Black H S. Photochem. Photobiol. 1987, 46, 213-221). Formation of UV-induced ROS causes oxidative damage to lipids, proteins and DNA (Vile G F and Tyrrell R M. Free Radic. Biol. Med, 1995, 18, 721-722; Chen Q, et al. Proc. Natl. Acad. Sci. USA, 1995, 92, 4337-4341).
Under normal circumstances, low levels of ROS are neutralized by skin's constitutive antioxidant defenses. However, research has shown that even sub-erythemal doses of UVR generates such an abundance of ROS that skin's own antioxidant defenses become overwhelmed, resulting in a build up of ROS that are free to cause oxidation, which contributes to acute (immunesuppression and photosensitivity disorders) and chronic (photoaging and skin cancer) forms of skin damage (Thiele J J, et al. J. Invest. Dermatol, 1998, 110(5), 756-761; Sander C. S, et al. J. Invest. Dermatol, 2002, 118 (4), 618-625; Thiele J. J: Skin Pharmacol. Appl. Skin Physiol, 2001, 14 (suppl. 1), 87-91; Sander C S, et al. International Soc. Dermatol, 2004, 43, 326-335). EP 1591104 (STADA Pharmaceuticals AG) describes the use of antioxidants in pharmaceutical formulations for protection against infrared radiation.
Antioxidants (Aox) function to neutralize ROS. If the right type and level are present within skin where ROS are being formed, Aox should be able to neutralize ROS before they can attack and oxidize other biomolecules. Accordingly, it would be useful to have a method to determine which topical applied antioxidants can be highly effective at neutralizing UVR-induced reactive oxygen species (ROS) within skin. Further, it would be useful to have a method to distinguish compounds which may only be effective in solution to scavenge free radicals from compounds that may be highly effective antioxidants on skin when exposed to UVR. Further, it would be useful to have a method to determine the correct choice and use-level of antioxidants in sunscreen products to provide extra protection against skin damage caused by UVR-induced ROS. In addition, it would be useful to have a composition that provides protection from UVR-induced ROS both at the skin surface and deep in the epidermis, for example as far as the basal layer. These and other objectives are provided by the invention described herein.
Accordingly the invention described herein provides, inter alia, a method which comprises two unique ex vivo methods to assess the ability of topically applied Aox to provide protection against UVR-induced ROS formation within skin's outer layers. The first method uses microscopy, e.g., fluorescence microscopy, to image and quantify ROS formation in the inner layers of the epidermis, e.g. through to the basal layers, by imaging sections of human skin. The second method quantifies the extent to which the Aox containing composition inhibits peroxidation of lipids in skin's outer layers. This specification also demonstrates that a commonly used laboratory test to measure efficacy of antioxidants in solution to scavenge free radicals is not predictive of an Aox's ability to function effectively on more complex biological substrates like skin exposed to UVR. Thus, the present invention provides an advantage over prior art methods to select Aox for use in sunscreen products to ensure they provide a protective benefit.
All patent and non-patent references cited herein are hereby incorporated in their entirety into this specification by reference thereto. Identification or discussion of any reference in this section or any part of this specification shall not be construed as an admission that such reference is available as prior art to the present application.